Notch, first identified in Drosophila, is the founding member of a family of transmembrane receptor proteins that mediate cell responses to intrinsic and/or extrinsic developmental cues. The cellular response to Notch signaling can be differentiation, proliferation and/or apoptosis depending on the specific developmental program. In addition to its role as a signal-transducing cell surface protein, Notch can exert its function by directly regulating gene transcription. The Notch signaling pathway comprises Notch proteins: Drosophila Notch, LIN-12 and GLP-1 in C. elegans and Notch 1-4 in mammals; ligands: Delta, Delta-1, Delta-like 1 and 3, Jagged 1 and Jagged 2 (Serrate 1 and 2 in Drosophila, respectively); intracellular effectors: CBF-1, Deltex and NF-kappa B; target genes: HES, bHLH and TLE; processing molecules: Kuzbanian; and modifiers: numb, numb-like and disheveled 1, 2 3, as well as fringe family members, lunatic fringe, manic fringe, radical fringe (Laufer et al., U.S. Pat. No. 6,054,298; Johnston, et al., (1997) Development 124: 2245-2254).
Structural conservation of Notch family members, ligands and modifiers are seen throughout phylogeny, suggesting a conserved role for this signaling pathway in various species. The product of the Delta gene, acting as a ligand, and that of the Notch gene, acting as a receptor, are key components in a lateral-inhibition signaling pathway that regulates the detailed patterning of many different tissues in Drosophila (Bray (1998) Semin Cell Dev Biol 9:591). In humans, it has been shown that the Notch3 gene, located on chromosome 19, is mutated in CADASIL (for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) patients (Joutel et al., (1996) Nature 383: 707-710). CADASIL causes a type of stroke and dementia whose key features include recurrent subcortical ischemic events, progressive vascular dementia, craniofacial paralysis, migraine and mood disorders with severe depression (Chabriat et al. (1995) Lancet 346: 934-939).
Defects in the Notch signaling pathway may also be involved in other neurological diseases. For example, the genes encoding the amyloid precursor proteins, presenilin 1 (PS1) and presenilin 2 (PS2), are homologous to gene that has been genetically linked to a Notch family receptor in C. elegans, and contain mutations which have been linked to Alzheimer's (Levitan and Greenwald (1995) Nature 377:351-354). Therefore, the Notch signaling pathway may be required for proper neurologic development and function.
In addition, analysis of gene expression patterns for Notch and its ligands has indicated that Notch signaling may have a role in hematopoiesis (Milner and Bigas (1999) Blood 93:2431). Notch activation can lead to expansion of early progenitor cells resident in the bone marrow and other sites of hematopoiesis (Milner et al. (1994) Blood 83:2057). Notch-1 was also shown to be involved in determination of T-cell education and fate in the thymus (Robey (1999) An Rev Immunol 17:283). Furthermore, a subset of human T-cell leukemia patients harbor a translocation involving the Notch 1 gene which results in a constitutively active Notch protein (Ellisen et al. (1991) Cell 66:649) and Notch1 gene translocations have been associated with a minority of T-cell lymphoblastic leukemias (Aster (1994) Cold Spring Harb. Symp. Quant. Biol. 59:125-136). Truncated Notch 2 sequences have been thought to play a role in the development of thymomas in cats infected with the feline leukemia virus (Rohn et al. (1996) J Virol 70:8071). Compelling evidence that the Notch signaling pathway is involved in B-cell development is seen in B-cell malignancies induced by Epstein-Barr virus (EBV). EBNA2, the transforming protein of EBV, transactivates cellular genes by direct interaction with a primary component of the Notch pathway (Henkel et al. (1994) Science 265:92).
The Notch signaling pathway members, therefore, have been implicated in a number of disease states including, for example, neurologic, vascular, immunological, and hematologic disorders, as well as various malignancies. Thus, Notch signaling pathway therapeutics are highly desirable for treating various diseases and disorders.
The Drosophila Notch protein encodes a glycosylated transmembrane receptor having a molecular mass 350 KD, which is involved in cell-fate specification during development (Wharton et al., (1985) Cell 43:567-581; Artavanis-Tsakonas et al., (1995) Science 268: 225-232). Based on analysis of Drosophila mutants, it is thought that Notch is a receptor having different functional domains, with the intracellular domain having the intrinsic signal-transducing activity of the intact protein and the extracellular domain a regulating activity (Rebay et al., (1993) Cell 74: 319-329).
It has recently been found that fringe proteins have glycosyltransferase activity which is required for effective in vivo fringe function. The Drosophila protein fringe (FNG) has been shown to be a glucosaminyltransferase that controls the response of the Notch receptor to specific ligands Delta/Serrate/Jagged through glycosylation (Blair (2000) Current Biology 10:R608-R612). FNG is localised to the Golgi apparatus, rather than secreted as previously believed (Munro and Freeman (2000) Current Biology 10:813-820).
Glycosyltransferases catalyze the synthesis of glycoconjugates, including, glycoproteins, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of the carbohydrate chain. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme (Amado et al. (1999) Biochim Biophys Acta 1473:35-53; Kapitonov et al. (1999) Glycobiology 9:961-78). Because the glycosylation reaction is highly specific with respect to both the configuration of the sugar residue and the site of the addition, it is expected that unique domain structures for substrate recognition and nucleotide-sugar binding are located within the enzyme molecule.
Glycosylation is the principal chemical modification to proteins as they pass through Golgi vesicles. Glycosyltransferases of the Golgi do not possess an obvious sequence homology which would suggest a common Golgi retention signal. However, they are all membrane proteins and share type II topology, consisting of an amino terminal cytoplasmic tail, a signal anchor transmembrane domain, a stem region, and a large luminal catalytic domain. The membrane-spanning domain and its flanking regions contain necessary and sufficient information for Golgi retention of these enzymes (Jaskiewicz (1997) Acta Biochim Pol 44:173-9).
Notch signaling pathway therapeutics are highly desirable for treating various diseases and disorders, including immunological, neurological and vascular disorders. More particularly, fringe therapeutics would be desirable in view of the modulatory role fringe proteins play in Notch signaling.